Methods for determining the presence or absence of microorganisms in lipid-containing compositions

Abstract

The present invention provides methods of validating the sterility of compositions which contain large quantities of lipids, and especially those lipid compounds which contain quantities of bacteriocidal agents such as antibiotics. The compositions may contain quantities of phospholipids in the form of liposomes, microcrystals, or microdroplets. The methods involve dissolving the lipid composition in a diluent solution, passing the solution through a filtration device, and then incubating microbes which may be captured by the filtration device to determine whether microorganisms are present in the composition.

What is claimed is:  
     
       1. A method of determining the presence or absence of microbes in a composition containing a lipid, comprising: 
       dissolving the composition containing a lipid in a diluent solution to form a diluted sample, the dissolving of the composition being performed by;  
       providing the diluent solution at a pH where the solubility of the composition containing a lipid or the solubility of a major component of the composition containing a lipid is enhanced relative to the solubility at other pH points, the diluent solution comprising one or more solubilizers; and  
       contacting the composition containing a lipid with the diluent solution;  
       passing the diluted sample through a filtration device;  
       incubating the microbes which may be present on the filtration device for a time necessary to observe the growth of microbes which may be present; and  
       examining the assay result to determine the presence or absence of microbes.  
     
     
       2. The method of  claim 1  wherein the diluent solution further comprises a salt. 
     
     
       3. The method of  claim 1 , wherein the composition containing a lipid contains liposomes, microdroplets, or microcrystals. 
     
     
       4. The method of  claim 1 , wherein the composition containing a lipid contains a phospholipid. 
     
     
       5. The method of  claim 4 , wherein the phospholipid is a lecithin. 
     
     
       6. The method of  claim 4  wherein the phospholipid is comprised in microcrystals, liposomes, or microdroplets. 
     
     
       7. The method of  claim 1  wherein the composition containing a lipid further comprises a bacteriocidal agent. 
     
     
       8. The method of  claim 7  wherein the bacteriocidal agent is an antibiotic. 
     
     
       9. The method of  claim 8  wherein the antibiotic comprises oxytetracycline. 
     
     
       10. The method of  claim 1  wherein the solubilizer comprises an emulsifier. 
     
     
       11. The method of  claim 1  wherein the solubilizer is an emulsifier of between four and eleven carbons. 
     
     
       12. The method of  claim 1  wherein the solubilizer is a polysorbate and/or sodium lauryl sulfate. 
     
     
       13. The method of  claim 12  wherein the polysorbate is polysorbate 20. 
     
     
       14. The method of  claim 1  further comprising a water soluble metal/halide salt. 
     
     
       15. The method of  claim 14  wherein the salt is sodium chloride or potassium chloride. 
     
     
       16. The method of  claim 1  wherein the one or more solubilizers comprise polysorbate 20 and/or sodium lauryl sulfate, and further comprising a water soluble metal/halogen salt. 
     
     
       17. A method of determining the presence or absence of microbes in a composition comprising an antibiotic suspended in a phospholipid matrix, comprising: 
       dissolving the composition in a diluent solution to form a diluted sample, the dissolution being performed by;  
       providing the diluent solution at a pH where the solubility of the composition or the solubility of a major component of the composition is enhanced relative to the solubility at other pH points, the diluent solution comprising one or more solubilizers and a salt;  
       contacting the composition with the diluent solution;  
       passing the diluted sample through a filtration device;  
       incubating the microbes which may be present on the filtration device for a time necessary to observe the growth of microbes which may be present;  
       examining the assay result to determine the presence or absence of microbes.  
     
     
       18. The method of  claim 17 , wherein the composition containing a lipid contains liposomes, microdroplets, or microcrystals. 
     
     
       19. The method of  claim 17 , wherein the composition containing a lipid contains a phospholipid. 
     
     
       20. The method of  claim 19 , wherein the phospholipid is a lecithin. 
     
     
       21. The method of  claim 19  wherein the phospholipid is comprised in microcrystals, liposomes, or microdroplets. 
     
     
       22. The method of  claim 17  wherein the antibiotic comprises oxytetracycline. 
     
     
       23. The method of  claim 17  wherein the solubilizer comprises an emulsifier. 
     
     
       24. The method of  claim 17  wherein the solubilizer is a polysorbate or sodium lauryl sulfate. 
     
     
       25. The method of  claim 17  wherein the solubilizer is an emulsifier of between 4 and 11 carbons. 
     
     
       26. The method of  claim 24  wherein the polysorbate is polysorbate 20. 
     
     
       27. The method of  claim 17  wherein the salt is a water soluble metal/halide salt. 
     
     
       28. The method of  claim 27  wherein the water soluble metal/halide salt is sodium chloride or potassium chloride.